Ectopic expression of WRINKLED1 in rice improves lipid biosynthesis but retards plant growth and development

WRINKLED1 (WRI1) is a transcription factor which is key to the regulation of seed oil biosynthesis in Arabidopsis. In the study, we identified two WRI1 genes in rice, named OsWRI1a and OsWRI1b, which share over 98% nucleotide similarity and are expressed only at very low levels in leaves and endosperms. The subcellular localization of Arabidopsis protoplasts showed that OsWRI1a encoded a nuclear localized protein. Overexpression of OsWRI1a under the control of the CaMV 35S promoter severely retarded plant growth and development in rice. Expressing the OsWRI1a gene under the control of the P1 promoter of Brittle2 (highly expressed in endosperm but low in leaves and roots) increased the oil content of both leaves and endosperms and upregulated the expression of several genes related to late glycolysis and fatty acid biosynthesis. However, the growth and development of the transgenic plants were also affected, with phenotypes including smaller plant size, later heading time, and fewer and lighter grains. The laminae (especially those of flag leaves) did not turn green and could not unroll normally. Thus, ectopic expression of OsWRI1a in rice enhances oil biosynthesis, but also leads to abnormal plant growth and development.


Introduction
Angiosperm seeds accumulate storage protein, oil (mainly triacylglycerols, TAG) and carbohydrate (mainly starches) during seed filling and degrade these compounds upon germination in order to support early seedling growth. The relative abundance of storage reserves varies among seeds of different species. Many oilseeds produce 50-70% oil, some legumes contain 40% protein, whereas the seeds of most cereals have starch as 70-85% of the dry weight [1]. The accumulation of storage components requires the coordinated expression of many genes that encode the enzymes of the corresponding pathways [2]. Several transcription factors have been reported to play roles in regulating these pathways. One of the most important of these genes is WRINKLED1 (WRI1) [3]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 The WRI1 gene products belong to the AINTEGUMENTA (ANT) subfamily in AP2/ERF transcription factor family, members of which contain two plant-specific (AP2/EREB) DNAbinding domains [4]. The function of the WRI1 gene in plants is first reported in Arabidopsis. Arabidopsis wri1 mutant seedlings have no obvious phenotype during vegetative development, but they produce wrinkled seeds and the ability of developing seeds to convert sucrose and glucose into precursors of fatty acid (FA) biosynthesis is impaired, subsequently causing an 80% reduction in seed oil content [3]. As a transcription factor, the WRI1 protein can target, and promote the expression of, many genes encoding enzymes involved in late glycolysis and FA biosynthetic pathways [5,6]. In Arabidopsis [4,7], Brassica napus [8,9], Nicotiana benthamiana [10], and Solanum tuberosum [11], overexpression or ectopic expression of WRI1 genes increases the oil content of seeds, leaves, or tubers.
In maize, overexpression of ZmWri1a increases the fatty acid content of the mature grain. The higher seed oil content in ZmWri1a overexpressing lines is not accompanied by any reduction in starch. The transgenic kernels do not show any macroscopically visible defects such as wrinkled or plump phenotypes, and there is no significant difference in kernel weight between transgenic and wild-type kernels [12,13]. The ectopic expression of BdWRI1 in the grass Brachypodium distachyon induces the transcription of genes predicted to be involved in glycolysis and FA biosynthesis, and TAG content is increased up to 32.5-fold in 8-week-old leaf blades. However, ectopic expression of BdWRI1 also cause cell death in leaves [14].
Rice (Oryza sativa L.) has two genes, LOC_Os11g03540 and LOC_Os12g03290, which are close to the Arabidopsis WRI1 gene on a phylogenetic tree [15]. The LOC_Os11g03540 gene (AJ575217) is found to be mainly expressed in embryos, however, the expression of this gene is hardly detected in leaves and endosperms [16]. Sun et al. [17] expresses the CoWRI1 gene of Coconut (Cocos nucifera L.) using an endosperm-specific promoter EnP2 from rice changes the seeds oil content in transgenic rice. In order to explore whether the expression of OsWRI1 could increase oil accumulation in non-oil storage organs such as endosperms and leaves of rice plants, we overexpressed the LOC_Os11g03540 gene under the control of the CaMV 35S promoter. We observed that the resulting transgenic plants were severely retarded with respect to growth and development. Expressing this gene under the control of the P1 promoter of Brit-tle2 (small subunit of ADP-glucose pyrophosphorylase) [18] increased the oil content of leaves and endosperm but affected plant growth and development in rice.

Plant materials
Using the japonica rice (Oryza sativa L.) cv. Zhonghua 11 (ZH11) as the wild type materials. After the seeds of wild-type and transgenic plants germinated, they were planted in a glass greenhouse under natural light in the South China Botanic Garden, China. Roots, leaves, embryos and endosperms from seeds of plants 10 days after flowering were sampled, and stored at −80˚C until required for expression profiles analysis.

Subcellular localization of OsWRI1a protein
Using rice leaves as a template, we cloned the OsWRI1a gene (without stop codon) by RT-PCR technology. Subsequently, we ligated the sequenced correct OsWRI1a gene into the pBWA(V) HS-GLosgfp vector to construct the OsWRI1a-GFP fusion protein vector. We further cotransformed the constructed OsWRI1a-GFP fusion expression vector and empty vector into Arabidopsis protoplast cells by a PEG-mediated method [20], and observed under a confocal electron microscope.

Plasmid constructs and plant transformation
Using rice leaf cDNA as template, specific primers were designed (primers given in S1 File), and the coding sequence of full-length OsWRI1 gene was cloned by RT-PCR. The cloned and purified DNA sequence was connected to the pMD18-T vector (Takara, http://www.takara. com.cn/). The qualified recombinant cloning vector pMD18-T -OsWRI1 plasmid was digested with BamH I and Sal I. Then, the DNA fragment cut by BamH I and Sal I was connected to pCAMBIA1301 vector under the control of the CAMV 35S promoter (35S) by T 4 DNA ligase (EcoR I -35S -Sac I, BamH I -OsWRI1a -Sal I, Pst Iocs -Hind III).
To construct an expression vector containing OsWRI1a under the control of the promoter of Brittle2 (Bt2, endosperm ADP-glucose pyrophosphorylase small subunit; LOC_Os08g25734), the P1 promoter of the Brittle2 gene was amplified by PCR from rice genomic DNA extracted from leaves (primers listed in S1 File) [18]. The correctly sequenced PCR product was connected to the pMD18-T vector, and the pMD18-T-OsWRI1 plasmid was digested by Hinc II and BamH I. Then we linked the target gene to the pCAMBIA1301 vector through T 4 DNA ligase (containing OsWRI1a and ocs) at the Sma I/BamH I site (Sma I/Hinc II -Bt2P1 -BamH I -OsWRI1a -Sal I, Pst Iocs -Hind III). Next, we transformed the constructed OsWRI1 plant expression vector into Agrobacterium (strain EHA105) by the freezethaw procedure. Finally, Agrobacterium containing OsWRI1 plasmid was used to transform rice callus as described by Li and Li [21,22], and obtained OsWRI1 transgenic plants. In addition, the screening of homozygotes was based on a segregation ratio of about 3:1 observed in T2 plants. The segregation ratios of four transgenic plants, were confirmed by GUS staining, were 237:78, 197:64, 169:55 and 206:67, respectively. Then we randomly selected 30 T3 plants for GUS staining. We speculated that these plants were homozygous plants for all GUS-stained plants.
TAG and free FA analyses were performed as described by previously Yang et al. [14]. Total lipid samples were separated by TLC on silica plates (Si250PA; 20 � 20cm; Mallinckrodt Baker) developed with ether: ethyl ether:acetic acid (80:20:1, v/v). After development, TAG and free FA bands were sprayed with 0.01% (v/v) Primuline in 80% (v/v) acetone and visualized under UV light. TAG or free FA bands were isolated from the TLC plate. FA methyl esters were prepared and quantified as described previously [26].
For fatty acid composition analysis. To determine fatty acid composition, fatty acid methyl esters were prepared and quantitatively analyzed by gas chromatography according to Wu et al. [27]. An Agilent 7890A GC system apparatus with a flame ionization detector on an HP-88 column (30 m × 0.25 mm I.D., 0.20 μm film thickness) was used. Results are expressed as mol percentage [26]. Statistical analysis of the data was performed using Student's test.

Results
The WRI1 genes of rice WRINKLED1 (WRI1) encodes a transcription factor of the AP2/ERF class. The protein has two plant-specific (AP2/ERF) DNA-binding domains, and in Arabidopsis it is involved in the control of storage compound biosynthesis [3,4]. According to a phylogenetic tree based on AP2/ERF family proteins from selected plant species, seven AP2 proteins from rice fall into the ANTb clade which contains the AtWRI1 protein [15] (Fig 1). LOC_Os11g03540 and LOC_Os12g03290 proteins share the highest amino acid similarities with Arabidopsis WRI1 and maize WRI1 proteins, so we named them OsWRI1a and OsWRI1b respectively. The coding domain sequences of the two OsWRI1 genes share very high (over 98%) nucleotide similarity. Their levels of expression could not be determined separately by RT-PCR. The expression levels measured by qRT-PCR in this study therefore represent the combined expression of the OsWRI1a and OsWRI1b genes, which were expressed mainly in roots and embryos (Fig 2). Of the two OsWRI1 genes, we could only obtain cDNA clones for OsWRI1a by RT-PCR from total RNA of young seeds. Therefore, the OsWRI1a gene was selected for further functional verification.

OsWRI1a localizes to the nucleus
In order to clarify the protein properties of OsWRI1a, we constructed an OsWRI1a-GFP fusion protein expression vector. Then we transferred the constructed vector and empty control vector into Arabidopsis thaliana protoplast cells by PEG-mediated method, and placed them under a fluorescence confocal microscope to observe the localization. The subcellular localization results showed that green fluorescence signal was detected in all cells with the empty control vector, while the fluorescence signal was only observed in the nucleus of the OsWRI1a-GFP fusion expression vector (Fig 3). The results show that the OsWRI1a protein is localized in the nucleus.

Overexpression of OsWRI1a in rice seriously retarded plant growth and development
Ectopic expression of the WRI1 gene in several plant species results in an increase in oil accumulation in leaves and other organs. To investigate whether ectopic expression of this gene exerted the same effect in rice, we overexpressed the WRI1a gene under the control of the CaMV 35S promoter. More than twenty transgenic lines were obtained. The transgenic seedlings exhibited multiple morphological defects, including dwarfism, chlorotic leaves, and failure to produce seeds. The upper parts of the laminae of most leaves were chlorotic and curly, and the chlorotic parts died quickly (S1 Fig). Because a high level of expression of the OsWRI1a gene seriously inhibited rice growth, and since expression of ZmWRI1 under the control of the 19 KD ZEIN promoter (expressed at the later stage of endosperm development) do not lead to an increase in seed oil content in maize [12], we next expressed the OsWRI1a gene under the control of the P1 promoter of the Brittle2 (Bt2) (LOC_Os08g25734) gene. The Bt2 gene encodes two proteins, a cytosolic and a plastidial small subunit of ADP-glucose pyrophosphorylase. The GUS gene under the control of the Bt2 P1 (P Bt2P1 ) is highly expressed in endosperm but lower in leaves and roots of rice plants, whereas under the control of the P2 promoter, it is expressed strongly in roots and leaves but at a low level in endosperms [18]. Four independent P Bt2P1 ::OsWRI1a transgenic lines (P1-6, -7, -8, and -10) were established for use in our experiments.

Expression of OsWRI1a in rice under the control of the Bt2P1 promoter affected plant growth and development
The phenotypes of P Bt2P1 ::OsWRI1a transgenic rice lines were observed in the T4 plants. It was found that P Bt2P1 ::OsWRI1a transgenic rice plants were smaller and their heading date was about 7 days later than the wild type (ZH11) (Fig 4A-4C). The number of tillers in P Bt2P1 ::

PLOS ONE
OsWRI1a plants (from 7.6±1.6 to 8.8±1.7) was less than that in the wild type (14.6±1.5) at the heading stage under present pot-culture conditions. The root lengths of P Bt2P1 ::OsWRI1a seedlings were about 5% less than the wild type (S2 Fig). The P Bt2P1 ::OsWRI1a plants had shorter internodes, shorter panicles and fewer grains than wild type plants (Fig 4E and 4F). The middle part of the lamina of all flag leaves and a small proportion of the second leaves did not turn green and failed to unroll properly during the period from emergence to death (Fig 5A-5D). Laminae of the second leaves were shorter, looked yellowish-green ( Fig 5E) and contained less chlorophyll compared to the leaves of the same part of the wild-type plants (Fig 5F). Changes in levels of OsWRI1a transcripts in leaves were observed by qRT-PCR analysis (Fig 4D). The levels of OsWRI1a transcripts in the first leaf (flag leaf) was higher than that in the second and third leaves at the flag leaf stage of about 10 cm in length in P Bt2P1 ::OsWRI1a transgenic plants ( S3A Fig). The crude lipid content of the leaves of P Bt2P1 ::OsWRI1a plants was increased by 40% to 65% (S3B Fig), and the expression of several genes involved in later glycolysis and fatty acid

PLOS ONE
The role of OsWRI1 gene in lipid biosynthesis biosynthetic pathway was upregulated (S3C Fig). To explore whether the levels of leaf TAG was also increased, we examined the content of TAG in the leaf blades of two P Bt2P1 ::OsWRI1a lines. As a result, the content of TAG in P Bt2P1 ::OsWRI1a leaf blades was increased by more

PLOS ONE
The role of OsWRI1 gene in lipid biosynthesis than 40% (Fig 5G). After analyzing the fatty acid composition, we observed that the TAG of the P Bt2P1 ::OsWRI1a lines contained more C18:1 and C18:2, but less C18:0 and C18:3 compared to wild-type lines (Fig 5I). Similar to the ectopic expression of BdWRI1 in B. distachyon [14], the total free fatty acid content and total unsaturated fatty acid content was also increased in P Bt2P1 ::OsWRI1a leaf blades (Fig 5G and 5H). These results indicate that the expression of OsWRI1a influences fatty acid biosynthesis in leaves and plant growth and development in rice.

The P Bt2P1 ::OsWRI1a lines had increased TAG content and altered fatty acid composition in endosperms
The 1000-seed weight of P Bt2P1 ::OsWRI1a transgenic rice plants was approximately 15% lower than those of wild type (S4A Fig). The total crude lipid content of the endosperm of the mature seeds from P Bt2P1 ::OsWRI1a lines was about 75% more than that of the wild type, while the net crude lipid content was no more than 1% (S4B Fig). We also analyzed the TAG content of endosperm and embryo of two P Bt2P1 ::OsWRI1a lines. The TAG content in the endosperm of mature seeds from P Bt2P1 ::OsWRI1a lines was over 30% more than that of the wild type ( Fig  6A). The C16:0, C18:0, C18:1, and C18:2 fatty acids were the main components of TAG of rice endosperm. The TAG of endosperm from P Bt2P1 ::OsWRI1a lines contained more C16:0, but less C18:1, compared to wild type (Fig 6B).

Expression of OsWRI1a upregulated the expression of glycolysis and fatty acid biosynthesis related genes in rice endosperm
In maize and many other plants, overexpression of the WRI1 gene upregulates the expression of many genes involved in glycolysis and in fatty acid biosynthetic pathways [6,8,12]. Therefore, we further examined the expression of several genes related to these pathways in the endosperm 5 and 10 days after flowering (DAF) by qRT-PCR. Compared to wild type, expression of 8 genes, PK, PDH-E1 α, PDH-E1β, PDH-E2, KASIII, ENR1, ACP, and FatA, was increased more than 2-fold at 5 DAF and/or 10 DAF (Fig 7).

Discussion
The WRI1 protein is a key regulator of oil biosynthesis first discovered in plants [3,28,29]. Mutation in the WRI1 gene causes a reduction in oil accumulation and wrinkled seeds. According to a phylogenetic analysis of AP2/ERF proteins, Arabidopsis has 3 AP2 proteins, while rice has 7 AP2 proteins that fall into the ANTb clade (Fig 1) [15]. As in the case of maize, the WRI1 gene is duplicated in rice. The OsWRI1 genes share very high nucleotide similarity, probably as a result of a recent segmental duplication between chromosomes 11 and 12 [30]. The OsWRI1 genes were barely expressed in leaves and endosperms (Fig 2) [16], but their expression was high in embryos, suggesting that they may play roles in the biosynthesis of storage fatty acids in rice embryos.
https://doi.org/10.1371/journal.pone.0267684.g005 expressing rice did not turn green and could not unroll. The growth and development of the transgenic seedlings were severely retarded (S1 Fig). In rice expressing P Bt2P1 ::OsWRI1a, growth and development were less strongly retarded. Laminae of the flag leaves did not turn green and failed to unroll (Fig 5A-5D). These effects should base on the abundance of OsWRI1 transcripts in different leaves of P Bt2P1 ::OsWRI1a transgenic lines (S3A Fig). These results implied that a higher expression level of the OsWRI1 gene could inhibit chlorophyll biosynthesis and photomorphogenesis in transgenic rice leaves, as was suggested in the case of partial Arabidopsis overexpressing WRI1 lines under the control of the 35S promoter [4]. The development of rice leaves may be more sensitively to this inhibitory effect than Arabidopsis because it appears in all 35S::OsWRI1a expressing rice plants. Previous studies suggest that overexpression of AtWRI1 in rice shows a significant increase in vegetative tissue fatty acids, with some seed reserve changes, but no phenotypic off types observed [31,32]. We speculate that the different phenotypes of AtWRI1-overexpressing rice plants and OsWRI1-overexpressing rice plants may be attributed to the fact that genes in different species may play different functions, even though these genes are capable of increasing fatty acid content and are highly homologous. Similarly, JcERF011 functions differently in Arabidopsis and rice [15]. In maize, overexpression of ZmWRI1a results in kernels that have elevated oil content, but do not show any visible defects with respect to seed yield and other agronomic traits [12,13]. In Brassica napus, overexpression of BnWRI1 accelerates flowering and enhances oil accumulation in both seeds and leaves without leading to any visible inhibition of growth [9]. Ectopic expression of BdWRI1 in the grass Brachypodium distachyon induces the transcription of genes predicted to be involved in glycolysis and FA biosynthesis, and TAG content is increased in leaf blades. However, ectopic expression of BdWRI1 also cause cell death in the leaf blades; this may be due to the high content of free fatty acids [14]. Taking these findings together, it appears that the specific phenotypes resulting from ectopic expression of the WRI1 gene depend on plant species even within the same family.
As in other plant species, the WRI1 gene has a function in FA biosynthesis in Arabidopsis and rice [8,12,33]. The expression of OsWRI1a under the control of the P1 promoter from the Bt2 gene was sufficient to increase the lipid content of rice leaves and endosperms (Figs 5G and 6A). The underlying mechanism involves the transcriptional up-regulation of genes related to late glycolysis and fatty acid biosynthesis in leaves and endosperms (S3C Fig; Fig 7). An unresolved question was why, although the oil content was increased, the net content in mature endosperms was no more than 1% (Fig 6A and S4B Fig). The same phenomenon was also observed in maize seeds as a result of overexpression of ZmWRI1a [12,13]. The first possible explanation is the cytosolic localization of ADP-glucose pyrophosphorylase in rice and maize endosperms, which is responsible for partitioning large amounts of carbon into starch [34]. Another possibility is that the WRI1 protein regulates mainly the expression of the genes involved in glycolysis and FA biosynthesis, not those of the TAG assembly pathway. The third possibility is that, in oilseeds, the accumulation of oils in endosperms or cotyledons accompanies the degradation of starch and the assembly of TAGs [2,26]. In contrast, TAGs seem to be rapidly turned over in leaves in B. distachyon plants overexpressing BdWRI1 [14]. Thus, ectopically expressing solely the OsWRI1 gene in rice, another grass species, would not be expected to result in the large-scale accumulation of TAG in leaves or endosperms.

Conclusion
In this study, we cloned the OsWRIa gene and analyzed its function in rice. The OsWRI1a gene encoded a nuclear localization protein. OsWRI1a-overexpressing plants not only increased oil content in transgenic rice leaves and endosperm, but also resulted in abnormal growth and development of transgenic plants. This result adds to our understanding of the AP2 family gene WRI1 in plant growth and development.